The closed circular form of the endogenous squirrel monkey type D retrovirus (SMRV) was molecularly cloned in bacteriophage LambdagtWES.LambdaB and designated LambdaSMRV. The restriction map of LambdaSMRV was determined and was found to be identical to the parental SMRV linear DNA except for the deletion of one long terminal repeat (LTR). The biological activity of LambdaSMRV was ascertained by DNA transfection of canine osteosarcoma D17 cells and the host range of the recovered virus was identical to that of parental SMRV. The LambdaSMRV restriction map was oriented to the viral RNA by using gene-specific probes from other retroviruses. Consistent with earlier studies, multiple copies of SMRV sequences were detected in squirrel monkey cellular DNA. Related DNA fragments were also detected in the DNAs of other primate species, including human. The genetic relationship between SMRV and other retroviruses was examined by Southern blotting and hybridization under low stringency. The pol regions of type A-related (m432), type B (MMTV) and avian type C (RSV and MAV) viruses were found to be related to SMRV pol by Southern hybridization and DNA sequence analysis. In contrast, these viruses did not share detectable homology with the pol genes of mammalian type C viruses. These findings establish the existence of two major pol gene families in the evolution of retroviruses. It was also possible to demonstrate the homology shared by the env genes of SMRV and mammalian type C viruses (SSAV, BaEV and R-MuLV. By heteroduplexing analysis, a 300-bp region of colinearity between the env genes of SMRV and BaEV was observed. These results indicate the genetic interaction between different retrovirus subfamilies has also played a role in the evolution of retroviruses.